Synthesis and conservation of ribosomal proteins during compensatory renal hypertrophy.
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ABSTRACT:
SUBMITTER: Melvin WT
PROVIDER: S-EPMC1162560 | biostudies-other | 1980 Apr
REPOSITORIES: biostudies-other
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Project description:The molecular mechanism underlying renal hypertrophy and progressive nephron damage remains poorly understood. Here we generated congenic ribosomal protein S6 (rpS6) knock-in mice expressing nonphosphorylatable rpS6 and found that uninephrectomy-induced renal hypertrophy was significantly blunted in these knock-in mice. Uninephrectomy-induced increases in cyclin D1 and decreases in cyclin E in the remaining kidney were attenuated in the knock-in mice compared with their wild-type littermates. Uninephrectomy induced rpS6 phosphorylation in the wild-type mice; however, no rpS6 phosphorylation was detected in uninephrectomized or sham-operated knock-in mice. Nonetheless, uninephrectomy stimulated comparable 4E-BP1 phosphorylation in both knock-in and wild-type mice, indicating that mTORC1 was still activated in the knock-in mice. Moreover, the mTORC1 inhibitor rapamycin prevented both rpS6 and 4E-BP1 phosphorylation, significantly blunted uninephrectomy-induced renal hypertrophy in wild-type mice, but did not prevent residual renal hypertrophy despite inhibiting 4E-BP1 phosphorylation in uninephrectomized knock-in mice. Thus, both genetic and pharmacological approaches unequivocally demonstrate that phosphorylated rpS6 is a downstream effector of the mTORC1-S6K1 signaling pathway mediating renal hypertrophy. Hence, rpS6 phosphorylation facilitates the increase in cyclin D1 and decrease in cyclin E1 that underlie the hypertrophic nature of uninephrectomy-induced kidney growth.
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Project description:During the first 48h of compensatory renal hypertrophy induced by unilateral nephrectomy, RNA content per cell increased by 20-40%. During this period, rates of RNA synthesis derived from the rates of labelling of UTP and RNA after a single injection of [5-(3)H]uridine showed no change in the rate of RNA synthesis (3.1nmol of UTP incorporated into RNA/min per mg of RNA). ATP and ADP pools were not changed. The rate of RNA synthesis was considerably in excess of the increment of total RNA appearing in the kidneys. With [5-(3)H]uridine as label, only continuous infusion for 24h could produce an increase (60%) in the specific radioactivity of renal rRNA in mice with contralateral nephrectomies. With a single injection of [methyl-(3)H]methionine used to identify methyl groups inserted into newly synthesized rRNA, the specific radioactivity of this rRNA was unchanged 5h after contralateral nephrectomy, increased by 60% at 9-48h, and returned to normal values at 120h. Most RNA synthesized in both nephrectomized and sham-nephrectomized mice has a short half-life. Since total cellular RNA content increases in compensatory hypertrophy despite unchanged rates of rRNA synthesis, the accretion of RNA might involve conservation of ribosomal precursor RNA or a change in rate of degradation of mature rRNA.
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Project description:The mechanism driving the remarkable ability of the remaining kidney to enlarge and increase its function following the removal of its contralateral pair remains elusive. To explore the pathways driving compensatory renal hypertrophy, comprehensive RNA-seq transcriptional studies were undertaken in the kidneys of C57BL/6 mice undergoing hypertrophy at 24, 48, and 72 hours following nephrectomy, and these results were compared with mice undergoing sham operations. In addition, mass spectrometry was carried out at 24 hours to examine changes in protein expression. Single-nuclei RNA-Seq was used to delineate bulk RNA-seq data into cell types at 24 hours post-nephrectomy. HK-2 renal tubular cells were examined for their ability to undergo hypertrophy in the presence of IGF-1 via the activation of cholesterol biosynthesis pathways. Bulk RNA-seq revealed substantial time-dependent enhancement of cholesterol biosynthesis pathways, increases in mitochondrial gene expression, and cell cycle perturbations. Single-nuclei RNA-Seq at 24 hours post-nephrectomy showed that Sterol Binding Protein 2 (SREBP2) activity increases in medullary thick ascending limb cells and, to a lesser extent, in proximal tubular cells, consistent with the role of promoting cholesterol synthesis. Furthermore, SREBP2 was found to regulate cell size following IGF-1 stimulation in HK-2 cells. There are early, cell-specific alterations in gene expression of cholesterol biosynthesis pathways, mitochondrial genes, and the cell cycle in kidneys undergoing compensatory hypertrophy. SREBP2 activity in the medullary thick ascending limb and, to a lesser extent, in proximal tubules may play a previously undescribed role in promoting cholesterol metabolism in the mechanism underlying compensatory renal hypertrophy.
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Project description:This mouse kidney outer cortex proteomics data set is part of multi-omic approach in a mouse unilateral nephrectomy model to identify signaling processes associated with compensatory hypertrophy of the renal proximal tubule.
2023-06-05 | PXD039697 | Pride
Project description:Loss of a kidney results in compensatory growth of the remaining kidney, a phenomenon of considerable clinical importance. However, the mechanisms involved are largely unknown. Here, we used a multi-omic approach in a mouse unilateral nephrectomy model to identify signaling processes associated with compensatory hypertrophy of the renal proximal tubule. Morphometry applied to microdissected proximal tubules showed that growth of the proximal tubule involves a marked, rapid increase in cell volume rather than cell number. Measurements of DNA accessibility (ATAC-seq), transcriptome (RNA-seq) and proteome (quantitative protein mass spectrometry) independently identified patterns of change that are indicative of activation of the lipid-regulated transcription factor, PPARα. Activation of PPARα by fenofibrate administration increased proximal tubule cell size, while genetic deletion of PPARα in mice decreased it. The results indicate that PPARα is an important determinant of proximal tubule cell size and is a likely mediator of compensatory proximal tubule hypertrophy.
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Project description:Coordinated functional balance of negative and positive transcription complexes maintain and accommodate gene expression in hearts during quiescent and hypertrophic conditions, respectively. Negative elongation factor (Nelf) complex has been implicated in RNA polymerase II (pol II) pausing, a widespread regulatory transcriptional phenomenon observed across the cardiac genome. Here, we examine the role of NelfA aka, Wolf-Hirschhorn syndrome candidate 2 (Whsc2), a critical component of the negative elongation complex in hearts undergoing pressure-overload induced hypertrophy. Alignment of high-resolution genome-wide occupancy data of NelfA, Pol II, TFIIB and H3k9ac from control and hypertrophied hearts reveal that NelfA associates with active gene promoters. High NelfA occupancy is seen at promoters of essential and cardiac-enriched genes, expressed under both quiescent and hypertrophic conditions. Conversely, de novo NelfA recruitment is observed at inducible gene promoters with pressure overload, accompanied by significant increase in expression of these genes with hypertrophy. Interestingly, change in promoter NelfA levels correlates with the transcript output in hypertrophied hearts compared to Sham, suggesting NelfA might be playing a critical role in the regulation of gene transcription during cardiac hypertrophy. In vivo knockdown of NelfA (siNelfA) in hearts subjected to pressure-overload results in early ventricular dilatation and dysfunction, associated with decrease in expression of inducible and cardiac-enriched genes in siNelfA hypertrophied compared to control hypertrophied hearts. In accordance, in vitro knockdown of NelfA in cardiomyocytes showed no change in promoter pol II, however significant decrease in in-gene and downstream pol II occupancy was observed. These data suggest an inhibited pol II progression in transcribing and inducible genes, which reflects as a decrease in transcript abundance of these genes. These results indicate that promoter NelfA occupancy is essential for pol II -dependent transcription. Therefore, we conclude that NelfA is required for active transcription and gene expression during cardiac hypertrophy.
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Project description:RNA chaperone activity is defined as the ability of proteins to either prevent RNA from misfolding or to open up misfolded RNA conformations. One-third of all large ribosomal subunit proteins from E. coli display this activity, with L1 exhibiting one of the highest activities. Here, we demonstrate via the use of in vitro trans- and cis-splicing assays that the RNA chaperone activity of L1 is conserved in all three domains of life. However, thermophilic archaeal L1 proteins do not display RNA chaperone activity under the experimental conditions tested here. Furthermore, L1 does not exhibit RNA chaperone activity when in complexes with its cognate rRNA or mRNA substrates. The evolutionary conservation of the RNA chaperone activity among L1 proteins suggests a functional requirement during ribosome assembly, at least in bacteria, mesophilic archaea and eukarya. Surprisingly, rather than facilitating catalysis, the thermophilic archaeal L1 protein from Methanococcus jannaschii (MjaL1) completely inhibits splicing of the group I thymidylate synthase intron from phage T4. Mutational analysis of MjaL1 excludes the possibility that the inhibitory effect is due to stronger RNA binding. To our knowledge, MjaL1 is the first example of a protein that inhibits group I intron splicing.
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Project description:Ribosome assembly occurs mainly in the nucleolus yet recent studies have revealed robust enrichment and translation of mRNAs encoding many ribosomal proteins (RPs) in axons, far away from neuronal cell bodies. Here, we report a physical and functional interaction between locally synthesized RPs and ribosomes in the axon. We show that axonal RP translation is regulated through a novel sequence motif, CUIC, that forms an RNA-loop structure in the region immediately upstream of the initiation codon. Using imaging and subcellular proteomics techniques, we show that RPs synthesized in axons join axonal ribosomes in a nucleolus-independent fashion. Inhibition of axonal CUIC-regulated RP translation causes a significant decline in local translation activity and markedly reduces axon branching in the brain, revealing the physiological relevance of axonal RP synthesis in vivo. These results suggest that axonal translation supplies cytoplasmic RPs to maintain/modify local ribosomal function far from the nucleolus in neurons.
2019-12-11 | GSE135502 | GEO
Project description:The mechanisms responsible for the increase in RNA per cell during the first 48h of renal compensatory growth were studied in the renal cortex. Unilaterally nephrectomized, sham-operated or non-operated rats were used. Incorporation into RNA of labelled precursors was studied in vivo and in vitro. Sham-operation produced significant changes in precursor incorporation, absolute amounts of UTP and RNA, and the rate of RNA synthesis. At 6h after surgery, the amount of RNA decreased in sham-operated controls, whereas that in growing cortex remained unchanged. Incorporation into RNA in vivo was greater in the growing cortex, although the rate of RNA synthesis was not increased. At 24h, precursor incorporation into RNA and UTP and RNA synthesis were all increased in the growing cortex. In contrast with results obtained in vivo, slices of growing cortex incorporated less labelled precursor into RNA than did cortex slices from sham-operated controls, from 3 to 48h. Maximal differences were found from 6 to 24h. An attempt was made to equalize endogenous precursor pool sizes by increasing the concentration of unlabelled uridine in the media; incorporation differences were narrowed significantly. Serum from nephrectomized animals did not increase precursor incorporation into RNA in vitro. An increase in RNA synthesis is an important factor in RNA accretion in the renal cortex beyond 12h of compensatory growth. This is accompanied by increased UTP content and preceded by expansion of other pools. The amount of labelled precursor incorporated into RNA is greatly influenced by its delivery rate to the growing kidney in vivo and by intracellular dilution of expanded precursor pools in vitro.
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